Biosensors provide an analysis of a biological fluid, such as whole blood, serum, plasma, urine, saliva, interstitial, or intracellular fluid. Typically, biosensors have a measurement device that analyzes a sample residing in a test sensor. The sample usually is in liquid form and may be a biological fluid or a derivative of a biological fluid, such as an extract, a dilution, a filtrate, or a reconstituted precipitate. The analysis performed by the biosensor determines the presence and/or concentration of one or more analytes in the biological fluid. Examples of analytes include alcohol, glucose, uric acid, lactate, cholesterol, bilirubin, free fatty acids, triglycerides, proteins, ketones, phenylalanine, or enzymes. The analysis may be useful in the diagnosis and treatment of physiological abnormalities. For example, a diabetic individual may use a biosensor to determine the glucose level in whole blood, and this information may be used in adjusting the individual's diet and/or medication.
Biosensors may be designed to analyze one or more analytes and may use different sample volumes. Some biosensors may analyze a single drop of whole blood, such as from 0.25-15 microliters (μL) in volume. Biosensors may be implemented using bench-top, portable, and like measurement devices. Portable measurement devices may be hand-held and allow for the identification and/or quantification of one or more analytes in a sample. Examples of portable measurement devices include the BREEZE® and CONTOUR® meters of Bayer HealthCare in Tarrytown, N.Y., while examples of bench-top measurement devices include the Electrochemical Workstation available from CH Instruments in Austin, Tex.
In electrochemical biosensors, the analyte concentration is determined from an electrical signal generated by an oxidation/reduction or redox reaction of the analyte, or of a species responsive to the analyte, when an input signal is applied to the sample. The input signal may be applied as a single electrical pulse or in multiple pulses, sequences, or cycles. A redox substance, such as a mediator, an enzyme or similar species, may be added to the sample to enhance the electron transfer from a first species to a second species during the redox reaction. The redox substance(s) may react with a single analyte, thus providing specificity to a portion of the generated output signal.
Electrochemical biosensors usually include a measurement device having electrical contacts that connect with electrical conductors in the test sensor. The test sensor may be adapted for use outside, inside, or partially inside a living organism. When used outside a living organism, a sample of the biological fluid is introduced into a sample reservoir in the test sensor. The test sensor may be placed in the measurement device before, after, or during the introduction of the sample for analysis. When inside or partially inside a living organism, the test sensor may be continually immersed in the sample, or the sample may be introduced intermittently to the test sensor. The test sensor may include a reservoir that partially isolates a volume of the sample, or the test sensor may be open to the sample. Similarly, the sample may continuously flow through the test sensor or be interrupted for analysis.
The test sensor may be formed by disposing or printing electrodes on an insulating base, such as by disposing one or more reagent compositions on one or more of the conductors. More than one of the conductors may be coated by the same reagent composition, such as when the working and counter electrodes are coated by the same composition. Multiple techniques known to those of ordinary skill in the art may be used to dispose the reagent composition on the test sensor. The reagent composition may be disposed on the conductors as a reagent fluid and then dried. When the sample is introduced to the test sensor, the reagent composition begins to rehydrate.
The reagent compositions disposed on each conductor may be the same or different. Thus, the reagent composition of the working electrode may contain an enzyme, a mediator and a binder, while the reagent composition of the counter electrode may contain only a mediator, which could be the same as or different from the mediator of the working electrode, and a binder. The reagent composition may include an ionizing agent for facilitating the oxidation or reduction of the analyte, such as an oxidoreductase enzyme, as well as any mediators or other substances that assist in transferring electrons between the analyte and the working electrode.
One or more components of a reagent composition may undergo a chemical transformation prior to use of the test sensor. In particular, it is believed that the oxidation state of the mediator may change over time under certain conditions. Mediators such as ferricyanide and organic quinones and hydroquinones may undergo reduction in the presence of water. The presence of reduced mediator in the reagent composition can cause an increase in background current of the sensor, leading to inaccurate and imprecise assay results, particularly for samples with low analyte concentration.
Typically, undesirable and/or premature chemical transformations in the reagent composition are inhibited by storing the test sensor in proximity to a desiccant. Desiccants typically are used in test sensor primary packaging, such as bottles or foil pouches, to prevent degradation of the reagent composition so as to maintain the desired shelf life of the test sensor. Conventional desiccants for test sensor storage systems can quickly adsorb moisture that may leak into the package containing the test sensor. Examples of desiccants used to protect test sensors include molecular sieves, such as those containing porous crystalline alumino-silicates, which quickly adsorb moisture even in low humidity environments.
A drawback to the protection of test sensors with a desiccant is that one or more components of the reagent composition may require a minimum level of moisture to retain their function in the composition. For example, the FAD dependent Glucose Dehydrogenase enzyme (FAD-GDH) is believed to require some residual moisture to maintain its native active configuration. Depletion of moisture from the reagent composition below a minimum level could lead to enzyme conformational change and inactivation of at least a portion of the FAD-GDH. Depletion of moisture from the reagent composition may result in inactivation of at least a portion of one or more other components of the reagent composition.
Loss of activity of an enzyme in the reagent composition due to excessive desiccation of the test sensor typically is addressed either by including excess amounts of enzyme in the reagent composition or by adding a substance to the reagent composition that is believed to stabilize the enzyme. Examples of substances that may stabilize the enzyme in a test sensor reagent composition include sugars such as trehalose or sucrose, and sugar alcohols such as mannitol, maltitol or sorbitol. These substances may be used in a lyophilization process to preserve enzyme activity. See, for example EP 1 785 483 A1. High loadings of the enzyme or of other substances such as stabilizers in the reagent composition can present other difficulties, however. Since the enzyme component typically is expensive, it is not desirable to increase the enzyme loading beyond the level needed for the assay. In addition, the enzyme or stabilizers can slow down the rehydration of the reagent composition by the sample, resulting in longer assay times, especially at lower temperatures. Excess enzyme in the test sensor, beyond that required for interaction with the analyte and/or an excess of other ingredients in the reagent composition such as the mediator, also may reduce the accuracy of the sensor.
Accordingly, there is an ongoing need for improved biosensor systems, especially those that may provide increasingly accurate and/or precise determination of the concentration of the analyte in the sample, and/or that may provide increasingly shorter analysis times. Moreover, there is a need for improved biosensor systems that have an increased shelf life over a wider range of storage conditions, while supplying the desired accuracy, precision and/or analysis time. The systems, devices, and methods of the present invention overcome at least one of the disadvantages associated with conventional biosensor systems.